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Lentivirus mediated RNA interference inhibits mesothelin expression in ovarian cancer cells and cell proliferation
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Abstract:
To construct a recombinant lentivirus plasmid of RNA interference targeting ( MSLN ) gene and to observe its effect on MSLN expression in human ovarian cancer cell line OVCAR 3 and its effect on cell proliferation. Methods: According to the Genbank information of MSLN, four RNA interfering sequences and a negative sequence were designed and inserted into plasmid pRNAT U6.2/Lenti and 5 kinds of plasmids were packaged: LV MSLN negative,LV MSLN shRNA1, LV MSLN shRNA2, LV MSLN shRNA3, and LV MSLN shRNA4; and they were used to transfect OVCAR 3 cells. Western blotting and indirect immunofluorescence were then used to investigate the interfering efficiency. The plasmid with high interfering efficiency was packaged. The cell proliferation test and clone forming test was used to assess the changes in cell proliferation. Results: DNA sequencing showed that the sequences of 5 recombinant lentivirus plasmids were correct. Lentivirus packaging was successfully done. Western blotting analysis confirmed that LV MSLN shRNA4 had the highest interfering efficiency (90%). MSLN specifically bound to cytomembrane of OVCAR 3 cells. Expression of MSLN in the interfered cells (OVC shRNA) was weaker than that in the control cells (OVC neg,OVC). OVC shRNA cells(\[11.2±1.3\]×10 5) grew slowly compared to OVC neg cells(\[20.5±2.5\]×10 5) and OVC cells(\[219±23\]×10 5) ( P ＜005）. There was a significant reduction in clone forming rate of OVC shRNA cells (152±2.1)% in comparison with OVC neg cells（\[27.9±2.5\]%) and OVC cells（\[288±31\]%）（ P ＜0.05）.Conclusions: We have successfully constructed MSLN RNAi recombination plasmid LV MSLN shRNA4, which can effectively inhibit MSLN expression and cell proliferation, which paves a way for studying MSLN function and gene therapy.
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Project Supported:
Supported by the National Natural Science Foundation of China（No.30572093）; the Natural Science Foundation of Hebei Province（No.C2005000804）
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